JP2008122725A - Confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal microscope unit applicable to an optional CCD camera, in which a banding noise is eliminated by completely synchronizing the scanning period of a Nipkow disk and the image capture period of the camera. <P>SOLUTION: The confocal microscope unit comprises a microscope unit and a confocal scanner unit having the Nipkow disk which rotates at a predetermined scanning period, and picks up a confocal image with the camera equipped with a frame transfer CCD element, the confocal image being obtained by focusing returning light of measurement light with which a specimen is irradiated, wherein a disk scanning period generation means is provided which generates the scanning period of the Nipkow disk having a period (T2=T1/n) which is the 1/n (integer n≥1) of the exposure time on the basis of the set exposure time (T1) of the CCD element. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention includes a confocal scanner unit having a microscope unit and a confocal scanner unit having a nipou disk that rotates at a predetermined scanning cycle, and forms a confocal image formed by imaging a return fluorescence of measurement light irradiated on a sample. The present invention relates to a confocal microscope for imaging with a camera including an element.

  A confocal microscope observes a sample by scanning a condensing point on the sample with a laser beam (hereinafter referred to as measurement light), imaging the return fluorescence from the sample, and obtaining an image. It is used for observation of physiological reactions and morphology of living cells in such fields as LSI surface observation in the semiconductor market.

  FIG. 5 is a functional block diagram showing a configuration example of a conventional confocal microscope. The confocal scanner unit 1 is connected to a port of the microscope unit 2. The measurement light L is condensed into an individual excitation light beam L1 by the microlens on which the microlens array disk 11 of the confocal scanner unit 1 is formed, passes through the dichroic mirror 12, and then the pinhole disk (hereinafter referred to as a "Nipou disk") 13. The light passes through each pinhole and is focused on the sample 4 on the stage 3 by the objective lens 21 of the microscope unit 2.

  By irradiating the excitation light beam L1, the sample 4 is fluorescent. The return fluorescence L2 emitted from the sample 4 passes through the objective lens 21 again and is collected on each pinhole of the Niipou disc 13. The return fluorescence that has passed through each pinhole is reflected by the dichroic mirror 12 and is imaged on the light receiving surface of the CCD element 51 provided in the camera 5 via the relay lens 14.

  The microlens array disk 11 and the tip disk 13 are coaxially coupled and are rotated at a predetermined speed by a motor 16 whose rotation is controlled by a motor driver 15. The condensing point on the sample 4 is scanned by the movement of the pinhole on the Nipo disk 13 by this rotation.

  Since the surface where the pinholes of the Niipou disc 13 are arranged, the surface to be observed of the sample 4 and the light receiving surface of the CCD element 51 are arranged in an optically conjugate relationship with each other, the optical element of the sample 4 is placed in the CCD element 51. A cross-sectional image, that is, a confocal image is formed. The details of the Niipou disc type confocal microscope are disclosed in Patent Document 1.

  In the confocal microscope having such a functional configuration, in order to remove striped noise (banding noise) included in the confocal image caused by uneven rotation of the Niipou disc, the rotational scan cycle of the Niipou disc and the imaging cycle of the camera are set. Need to be consistent.

  The matching method disclosed in Patent Document 2 will be described with reference to FIG. The scanning trigger signal of the Niipou disc 13 generated by the operation trigger generating circuit 6 that inputs the detection signal of the rotation sensor 17 of the Niipou disc 13 is guided to the imaging trigger supply unit 7.

  The imaging trigger supply unit 7 generates an imaging trigger signal in consideration of the scanning trigger signal and the vertical transfer time of the CCD element 51 and passes it to the imaging control unit 8, and the imaging control unit 8 generates a confocal image based on the imaging trigger signal. The imaging cycle of the camera 5 to be acquired is controlled.

  FIG. 6 is a time chart showing the relationship between the scanning trigger cycle T1, the vertical transfer time T2, the readout time T3, the exposure time T4 (= T1), and the imaging trigger cycle T5. The feature of this method is that the exposure time T4 coincides with the scanning trigger period T1, and the imaging trigger period T5 is the sum of the scanning trigger period T1 and the vertical transfer time T2.

Japanese Patent Laid-Open No. 5-60980 JP 2006-145634 A

  In order to eliminate banding noise, it is necessary to completely synchronize the imaging trigger of the camera and the scanning trigger of the Niipou disk. However, in the method disclosed in Patent Document 2, since the camera image capture cycle is simply the sum of the exposure time (= scanning trigger cycle) and the CCD vertical transfer time, the camera capture cycle and the Nipkow disc scanning cycle are used. The vertical transfer time is shifted, and the two cannot be synchronized at all.

  On the other hand, a general CCD camera has an imaging trigger terminal for receiving the capture timing from the outside, and has a function of performing exposure and transfer in synchronization with the external trigger. The Nipkow disc has a function of scanning in synchronization with an external pulse signal.

  When the conventional technique disclosed in Patent Document 2 is applied to a general CCD camera, the CCD imaging timing has priority when trying to synchronize the CCD imaging timing and the Nipo disk scanning from a single synchronization signal source. On the contrary, the CCD exposure time and the Nipo disk scanning period are shifted by the CCD vertical transfer time, resulting in uneven exposure (uneven light intensity) and banding noise. Therefore, the application of the prior art is limited to special cameras.

  The present invention has been made to solve the above-described problems, and can be applied to any CCD camera. Banding noise can be removed by completely synchronizing the scanning period of the Niipou disk and the image capturing period of the camera. The purpose is to realize a confocal microscope.

In order to achieve such a subject, the present invention has the following configuration.
(1) A confocal image composed of a microscope unit and a confocal scanner unit having a Nipkow disk that rotates at a predetermined scanning cycle, and which forms an image of return light of measurement light irradiated on a sample, is converted into a frame transfer CCD element. In a confocal microscope that images with a camera equipped with
Based on the set exposure time (T1) of the CCD element, a disk scan that generates a scan period of the Nipkow disk having a period (T2 = T1 / n) that is an integer (n ≧ 1) of the exposure time. A confocal microscope comprising a period generation unit.

(2) Acquisition of a camera for acquiring the scanning period (T2) from the disk scanning period generation means and generating an adjustment time (T4 = T1-T3) obtained by calculating a difference from the vertical transfer time (T3) of the CCD element The confocal microscope according to (1), further comprising a period generation unit.

(3) The camera capture cycle generation means generates a camera capture cycle (T5 = T1 + T3 + T4) obtained by calculating a sum of the exposure time (T1), the vertical transfer time (T3), and the adjustment time (T4). The confocal microscope according to (1) or (2), wherein

(4) Any one of (1) to (3), further comprising a synchronization control unit that acquires the camera capture period (T5) and passes a camera imaging trigger signal having a frequency that is the reciprocal of the period to the camera. A confocal microscope according to any one of the above.

(5) The synchronization control unit passes a disk trigger signal having a frequency obtained by multiplying the frequency of the camera imaging trigger signal by (n + 1) to a motor driver that controls the rotation of the Niipou disk. Confocal microscope.

According to the present invention, the following effects can be expected.
(1) In the present invention, by providing an adjustment time in consideration of the vertical transfer time of the CCD element, it is possible to completely synchronize the image capture timing of the camera and the scan timing of the Niipou disc with a single frequency trigger. Banding noise caused by uneven rotation of Niipou disc can be eliminated.

(2) Since an external synchronization terminal provided in a general CCD camera can be used as it is, it can be widely applied regardless of the camera.

  The present invention will be described in detail below with reference to the drawings. FIG. 1 is a functional block diagram showing an embodiment of a confocal microscope to which the present invention is applied. The same elements as those of the conventional configuration described with reference to FIG. Hereinafter, the characteristic part of the present invention will be described.

  In the case of actual live cell observation, in order to obtain an image with appropriate brightness, the user first sets conditions such as the exposure time of the camera.

  In the trigger generation unit 100, the disk scanning cycle generation means 101 is based on the exposure time (T1) of the CCD element 51 set by the user, and a cycle (T2 = 1) of an integer (n ≧ 1) of the exposure time. Generate a scanning period of the Niipou disc with T1 / n).

  The camera capture cycle generation means 102 obtains the Nipo disk scanning cycle (T2) from the disk scanning cycle generation means 101, and calculates the difference from the vertical transfer time (T3) of the CCD element 51 (T4 = T1-T3). ) Is generated.

  Further, the camera capture cycle generation means 102 generates a camera capture cycle having a cycle (T5 = T1 + T3 + T4) obtained by calculating the sum of the exposure time (T1), the vertical transfer time (T3), and the adjustment time (T4).

  The synchronization control unit 200 acquires a camera capture period (T5) from the trigger generation unit 100, generates a camera imaging trigger signal f1 having a frequency that is the reciprocal of the period T5, and passes it to the camera 5.

  Further, the synchronization control unit 200 acquires an integer n from the trigger generation unit 100, generates a disk trigger signal having a frequency f2 obtained by multiplying the frequency f1 of the camera imaging trigger signal by (n + 1), and controls the rotation of the Nipkow disk. It is passed to the driver 15.

  FIG. 2 is a time chart for explaining the operation of an embodiment of a confocal microscope to which the present invention is applied. In this embodiment, the case where the integer n is selected as n = 1 is shown. FIG. 2A shows a user-set exposure time T1 that occurs in synchronism with each camera imaging trigger period of FIG.

  FIG. 2B shows the disk scanning trigger period T2, and since n = 1, it coincides with the exposure time T1. FIG. 2C shows a vertical transfer time T3 of the CCD element that occurs every time the exposure time T1 ends.

  FIG. 2D shows an adjustment time T4 generated every time the vertical transfer time T3 ends, and is generated by calculating the difference between the disk scanning trigger period T2 and the vertical transfer time T3. FIG. 2E shows a camera imaging trigger period T5, which is generated by calculating the sum of the exposure time T1, the vertical transfer time T3, and the adjustment time T4.

  In the present invention, it is possible to completely synchronize the camera imaging trigger signal f1 and the disk scanning trigger signal f2 by setting the disk scanning trigger period T2 to 1 / integer of the exposure time T1 and providing the adjustment time T4. It is.

  The frequency of the disk scanning trigger signal f2 is a frequency obtained by multiplying the frequency of the camera imaging trigger signal f1 by (n + 1). In this embodiment, since n = 1, f2 is a frequency obtained by multiplying f1 by 2.

  FIG. 3 is a time chart for explaining the operation of another embodiment of the confocal microscope to which the present invention is applied. The feature of this embodiment shows a case where the integer n is selected as n = 3. The difference from FIG. 2 is that the disk trigger scanning period T2 in FIG. 3B is 1/3 of the exposure time T1.

  In this embodiment, since n = 3, f2 has a frequency obtained by multiplying f1 by four. In this embodiment, the scanning frequency of the Nipkow disk is increased, the rotation speed is increased, and the excitation light scans the sample three times during one exposure time, so that scanning is averaged and banding due to eccentricity of the disk Noise can be reduced.

  FIG. 4 is a time chart for explaining the operation of another embodiment of the confocal microscope to which the present invention is applied. In this embodiment, the case where the vertical transfer time T3 exceeds the scan trigger period T2 of the Niipou disc is shown. The trigger timing is essentially the same as in FIG.

It is a functional block diagram which shows one Embodiment of the confocal microscope to which this invention is applied. It is a time chart explaining operation | movement of one Embodiment of the confocal microscope to which this invention was applied. It is a time chart explaining operation | movement of other embodiment of the confocal microscope to which this invention was applied. It is a time chart explaining operation | movement of other embodiment of the confocal microscope to which this invention was applied. It is a functional block diagram which shows the structural example of the conventional confocal microscope. It is a time chart which shows the relationship between a scanning trigger period, a vertical transfer time, a readout time, an exposure time, and an imaging trigger period.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Confocal scanner unit 11 Micro lens array disk 12 Dichroic mirror 13 Nipou disk 14 Relay lens 15 Motor driver 16 Motor 2 Microscope unit 21 Objective lens 3 Stage 4 Sample 5 Camera 51 CCD element 100 Trigger generation part 101 Disk scanning period generation means 102 Camera Capture period generation means 200 synchronization control unit

Claims (5)

A camera comprising a microscope unit and a confocal scanner unit having a Nipkow disk that rotates at a predetermined scanning period, and a confocal image obtained by forming an image of the return light of the measurement light irradiated on the sample, a frame transfer CCD element In the confocal microscope for imaging by
Based on the set exposure time (T1) of the CCD element, a disk scan that generates a scan period of the Nipkow disk having a period (T2 = T1 / n) that is an integer (n ≧ 1) of the exposure time. A confocal microscope comprising a period generation unit.   Camera capturing period generating means for acquiring the scanning period (T2) from the disk scanning period generating means and generating an adjustment time (T4 = T1-T3) obtained by calculating a difference from the vertical transfer time (T3) of the CCD element. The confocal microscope according to claim 1, further comprising:   The camera capture cycle generation means generates a camera capture cycle (T5 = T1 + T3 + T4) obtained by calculating a sum of the exposure time (T1), the vertical transfer time (T3), and the adjustment time (T4). The confocal microscope according to claim 1 or 2.   The shared control unit according to any one of claims 1 to 3, further comprising a synchronization control unit that acquires the camera capture period (T5) and passes a camera imaging trigger signal having a frequency that is the reciprocal of the period to the camera. Focus microscope.   5. The confocal system according to claim 4, wherein the synchronization control unit passes a disk trigger signal having a frequency obtained by multiplying a frequency of the camera imaging trigger signal by (n + 1) to a motor driver that controls rotation of the Niipou disk. microscope.

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